Abstract The majority of chronic bacterial infections have been attributed to biofilm formation. Biofilm formation is a conserved physiological process during which bacteria become sessile, secrete a protective extracellular matrixandfunctionasacommunity,ratherthanassinglecells.Itisanadaptationmechanism,whichstarts with environmental cues that are transduced via cell signaling pathways and ultimately translated into changes in cellular behavior. The dinucleotide second messenger c-di-GMP, together with the enzymes for itsproductionanddegradation,hasbeenidentifiedasthemajorintracellularsignalingmoleculethatcontrols biofilmformationandvirulenceinmanybacterialspecies.Manymicrobesencodealargenumberofenzymes involved in c-di-GMP metabolism and receptors for c-di-GMP-dependent responses, and this number often scaleswiththeadaptationpotentialoftheorganism.Theprevalenceandorganizationofc-di-GMPsignaling networks suggests that mechanisms exist to ensure signaling specificity, although this hypothesis has not beenexploredingreatdetail.Here,studieswillfocusontheregulationofaconservedsignalingnetworkthat controlscelladhesioninawiderangeofbacteria,includingseveralmajorhumanpathogens.Centraltothis regulatory node is a transmembrane c-di-GMP receptor with a prevalent domain organization and the enzymes that control its activity. Preliminary data indicate that this system is ideal to study a major open questioninthefield:Howisc-di-GMPsignalingspecificityachievedinsignalingnetworkscontainingdozens of proteins with identical catalytic activities? We address this question several ways by focusing on the conserved, membrane-bound, HAMP domain-containing c-di-GMP receptor LapD. We explore how protein- protein interactions between this receptor and c-di-GMP metabolizing enzymes help confer specificity. Throughthesestudieswealsoaddresshowc-di-GMPsignalingiscontrolledacrossthecellmembrane,and how this protein family, comprised of >2000 HAMP-GGDEF-EAL domain-containing proteins, is regulated. Finally, we utilize mass spectroscopy-based proteomics approaches to systematically identify c-di-GMP- relevantproteinnetworks.Thesestudieswillbecomplementedbytheelucidationofspecificresponsesand signaling networks responsive to physiological inputs, foremost nutritional sources. Together, the proposed studieshavethepotentialtorevealbroadlyrelevantmolecularmechanismsthatarefundamentaltoc-di-GMP signaling and biofilm formation. Considering the central role of this process in infectious diseases, it is well accepted that understanding the underlying mechanisms may enable the development of innovative strategies to manage and treat chronic infections. Thus, the work described here will provide molecular blueprintsthatcanbeusedinthedesignofnew,targetedtherapies.